Protein-polymer hybrids have revolutionized the treatment of disease [Chemical Reviews, 2009, 109, 5402-5436; Nat Rev Drug Discov, 2003, 2, 347-360] and biocatalytic processes. [J. Am. Chem. Soc., 2006, 128, 11008-11009]. Protein-polymer hybrids typically comprise linear or branched polymers “grafted to” or “grafted from” accessable sites within the desired protein. These protein-polymer hybrids have already shown an impressive range of altered or improved properties. From a therapeutic perspective, the advantages of protein-polymer hybrids over native proteins include increased in vivo stability, minimized immune recognition due to steric effects, enhanced in vivo circulation, and improved therapeutic effects. Protein-polymer hybrids have also shown an increased solubility in non-aqueous media, which have expanded the utility of enzymatic biocatalytic processes into the realm of organic synthesis. [Biomacromolecules, 2009, 10, 1612-1618; Biotechnology Progress, 1994, 10, 398-402]
Recently, the concept of protein-polymer nanogel hybrids has been introduced in order to overcome some of the long-term stability issues associated with protein-polymer hybrids. [J. Am. Chem. Soc. 2006, 128, 11008-11009; J. Phys. Chem. B, 2008, 112, 14319-14324; J. Biotechnology 2007, 128, 597-605.] Some of these issues include organic solvent solubility and deactivation of traditional protein-polymer hybrids under harsh conditions. Both of these characteristics are extremely important for expanding the catalytic potential of enzymatic systems. Encapsulation of proteins into nanogel matrices have demonstrated superior temperature and organic solvent stability for several systems, such as carbonic anhydrase, lipase, and horseradish peroxidase among others. [Biomacromolecules, 2007, 8, 560-565 and 2009, 10, 1612-1618; J. Am. Chem. Soc. 2006, 128, 11008-11009; Angew. Chem., Int. Ed, 2008, 47, 6263-6266]
Traditionally, protein-polymer hybrids are synthesized in a two-step process. The proteins are first functionalized with N-hydroxysuccinimide-acrylate and then copolymerized with an acrylamide and a crosslinker using REDOX initiated free radical polymerization. However, this process produces uncontrolled, non-specific acrylate functionalization of the protein, and often leads to batch-to-batch variability of protein activity. This variability often originates from non-specific modification of lysine residues by the acrylate chemistry, resulting in deactivation of active sites and protein denaturing. [Nat Rev Drug Discov, 2003, 2, 214-221] Additionally, the polymers accessible through REDOX initiated free radical polymerizations are limited by a number of factors, including monomer selection, particle size, protein loading, and potential for controlled release properties.
More recently, protein-polymer hybrids have been prepared using controlled radical polymerization techniques (see Wang et al., Am. Chem. Soc. 1995, 117, 5614; Matyjaszewski & Xia, Chem. Rev. 2001, 101, 2921 (“Xie”); Matyjaszewski &Tsarevsky, Nature Chem. 2009, 1, 276) which allow unprecedented control over polymer dimensions (molecular weight), uniformity (polydispersity), topology (geometry), composition and chemical functionality. [Matyjaszewski, K., Ed, Controlled Radical Polymerization; ACS: Washington, D.C., 1998; ACS Symposium Series 685. Matyjaszewski, K., Ed.; Controlled/living Radical Polymerization. Progress in ATRP, NMP, and RAFT; ACS: Washington, D.C., 2000; ACS Symposium Series 768; Matyjaszewski, K., Davis, T. P., Eds. Handbook of Radical Polymerization; Wiley: Hoboken, 2002; Qiu, J.; Charleux, B.; Matyjaszewski, K. Prog. Polym. Sci. 2001, 26, 2083; Davis, K. A.; Matyjaszewski, K. Adv. Polym. Sci. 2002, 159, 1.]
While controlled radical polymerization techniques permit greater control over the polymer's composition, there is still a need for methods to attach those polymers to site-specific locations on a protein. Thus far, methods for site-specific incorporation of polymerization initiators into proteins have been limited to the N-terminal position or specific natural amino-acid directed linkages. Both of these suffer from challenging purification of intermediates and/or the inability to efficiently control the number or location of potential polymer connections, both of which can compromise the structural integrity of the modified protein.
While the many experiments conducted using in situ functionalized natural amino acids on proteins have illustrated the potential immense impact of well-defined protein-polymer hybrids, their application is limited by technical shortcomings, and there is a need to develop protein polymer hybrids where a desired polymer can be attached at a site-specific location on the protein. [See Broyer et al., J. Am. Chem. Soc. 2008, 130, 1041]